RNA Seq analysis of the role of calcium chloride stress and electron transport in mitochondria for malachite green decolorization by Aspergillus niger

Aspergillus niger was previously demonstrated to decolorize the commercial dye malachite green (MG) and this process was enhanced under calcium chloride (CaCl₂) treatment. Previous data also suggested that the decolorization process is related to mitochondrial cytochrome c. In the current work, we analyzed in depth the specific relationship between CaCl₂ treatment and MG decolorization. Gene expression analysis (RNA Seq) using Next Generation Sequencing (NGS) revealed up-regulation of 28 genes that are directly or indirectly associated with stress response functions as early as 30 min of CaCl₂ treatment; these data further strengthen our previous findings that CaCl₂ treatment induces a stress response in A. niger which enhances the ability to decolorize MG. A significant increase in fluorescence observed by MitoTracker dye suggests that CaCl₂ treatment also increased mitochondrial membrane potential. Isolated mitochondrial membrane protein fractions obtained from A. niger grown under standard growth conditions decolorized MG in the presence of NADH and decolorization was enhanced in samples isolated from CaCl₂-treated A. niger cultures. Treatment of whole mitochondrial fraction with KCN which inhibits electron transport by cytochrome c oxidase and Triton-X 100 which disrupts mitochondrial membrane integrity suggests that cyanide sensitive cytochrome c oxidase activity is a key biochemical step in MG decolorization. This suggestion was confirmed by the addition of palladium α-lipoic acid complex (PLAC) which resulted in an initial increase in decolorization. Although the role of cytochrome c and cytochrome c oxidase was confirmed at the biochemical level, changes in levels of transcripts encoding these enzymes after CaCl₂ treatment were not found to be statistically significant in RNA Seq analysis. These data suggest that the regulation of cytochrome c enzymes occur predominantly at the post-transcriptional level under CaCl₂ stress. Thus, using global transcriptomics and biochemical approaches, our study provides a molecular association between fungal mitochondrial electron transfer systems and MG decolorization.